Question

How does western blot western blot is accomplished technically.

Answer 1

Principle : Western blotting is a immunoblotting technique that rely on the specificity of binding between moleculeof interest and probe to allow detection of molecule of interest in the mixture of many other similar molecule. The molecule of interest is protein and the probe is antibody raised against that particular protein.

The SDS- PAGE technique is a prerequisite for western blot.

Procedure:

• The Tissue preperation:

The sample from the cell culture or from whole tissue has been taken and tissue are first broken using blender mechanically.

Assorted detergents, salts and buffers may be used for lysis of cell and to solublize protein. Tissue preparation is done often at cold temp. to avoid protein denature.

• Gel electrophoresis:

The protein samples are then separated by this process, may be by isoelectric point, molecular weight, electric charge, or a combination of these. The principle involved is the difference in electrophoretic mobilities of different proteins.

• Transfering :

In order to make proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose (NC) or polyvinylidene difluoride (PVDF).

The membrane is placed on the top of the gel and stack of filter paper placed on that. The entire stack is placed in a buffer solution which moves up the filter paper by capilary action, bringing the protein with it.

Another method is electrobloting for transferring using an electric current to pull protein from the gel into PVDF or NC. Protein binding is based upon hydrophobic interaction as well as charged interaction between membrane and protein. The uniformity of the trnsfer is checked by staining the membrane with Ponceau S dye.

• Blocking :

The both target and antibody are protein and the membrane have binding ability to protein, so there could be some unwanted binding. Blocking is achieved by placing the membrane in the solution of typically 3–5% bovine serum albumin (BSA) or non-fat dry milk (both are inexpensive) in tris-buffered saline (TBS) or I-Block.

The protein in the dilute solution attaches to membrane in all places where the target protein have not attached, thus when antibody is added there is no room on the membrane for it to attack other than on the binding site of specific target protein.

• Detection :

The membrane is probed for the protein of interest with modified antibody which is linked to a reporter enzyme, which when exposed to an appropriate substance derives a colorimetric reaction and produces a color.  

Detection can be done by colorimetric detection, radiometric detection or fluorescent detection.

  

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